Absence of selenoprotein P but not selenocysteine lyase results in severe neurological dysfunction.

1Cell and Molecular Biology Department, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, 96813, USA.

Abstract

Dietary selenium restriction in mammals causes bodily selenium to be preferentially retained in the brain relative to other organs. Almost all the known selenoproteins are found in brain, where expression is facilitated by selenocysteine (Sec)-laden selenoprotein P. The brain also expresses selenocysteine lyase (Scly), an enzyme that putatively salvages Sec and recycles the selenium for selenoprotein translation. We compared mice with a genetic deletion of Scly to selenoprotein P (Sepp1) knockout mice for similarity of neurological impairments and whether dietary selenium modulates these parameters. We report that Scly knockout mice do not display neurological dysfunction comparable to Sepp1 knockout mice. Feeding a low-selenium diet to Scly knockout mice revealed a mild spatial learning deficit without disrupting motor coordination. Additionally, we report that the neurological phenotype caused by the absence of Sepp1 is exacerbated in male vs. female mice. These findings indicate that Sec recycling via Scly becomes limiting under selenium deficiency and suggest the presence of a complementary mechanism for processing Sec. Our studies illuminate the interaction between Sepp1 and Scly in the distribution and turnover of body and brain selenium and emphasize the consideration of sex differences when studying selenium and selenoproteins in vertebrate biology.

Spontaneous activity and motor coordination is more reduced in male than female Sepp1−/− mice compared to control mice fed a standard diet. (A) Rearing and grooming were measured in the cylinder, and total distance traveled in the open field. Sepp1−/− mice were less active as measured by rearing (left, genotype **p<0.01) and grooming (center, genotype ***p<0.001), but did not show decreased exploration of the open field (right). (B) Motor coordination was measured using the pole test and inverted hang test. Sepp1−/− mice took longer to turn (left, genotype x sex ***p<0.001) and descend the pole (center, genotype x sex *p<0.05), and had reduced ability to suspend themselves for two minutes (right, genotype ***p<0.001). Values are expressed as means ± SEM. n=8–12 per group, *p<0.05, **p<0.01, ***p<0.001, compared with control mice.

High Se diet improves spontaneous activity and motor coordination more in male than female Sepp1−/− mice compared to control mice. (A) Rearing and grooming were measured in the cylinder, and total distance traveled in the open field. Se-supplemented Sepp1−/− mice were as active as controls when measured by rearing (left), grooming (center), and exploration of the open field (right), and no sex differences were observed. (B) Motor coordination was measured using the pole test and inverted hang test. Se-supplemented Sepp1−/− mice took longer to turn (left, genotype **p<0.01) and to descend the pole (center, genotype *p<0.05) when compared with control mice, but were capable of suspending themselves for two minutes (right). Male and female Sepp1−/− mice performed similarly except for turn time, and an interaction effect between genotype and sex was not statistically evident in any test. Values are expressed as means ± SEM. n=8–10 per group, ***p<0.001, compared with control mice.

Generation of Scly-knockout mice. (A) Image from KOMP. A promoterless trapping cassette was inserted upstream of exon 4 of the mouse Scly locus on chromosome 1, causing splicing at the cassette and truncation of the endogenous transcript. The cassette was flanked by FRT sites for conditional excision of the cassette by breeding with FLP-recombinase transgenic mice in case of embryonic lethality. Presence of the loxP sites flanking exon 4 allowed excision of a coding exon critical for enzymatic function by breeding with Cre-recombinase transgenic mice. (B) PCR genotyping of mice tails was performed to detect presence of wild-type allele (1.2 kb) or knockout allele (233 bp) using primers described in Materials and Methods. (C) Quantitative RT-PCR analysis of brains from Scly+/+ and Scly−/−mice indicated no detectable Scly mRNA in homozygous knockout mice.

Spontaneous activity and motor coordination is normal in male and female Scly−/− mice fed a low Se diet. (A) Rearing and total distance traveled was measured in the open field. Scly−/−mice were as active as controls when measured by exploration of the open field (right), however an interaction between genotype and sex affected rearing activity (left, genotype x sex *p<0.05). (B) Motor coordination was measured using the pole test. Scly−/− mice and control mice performed similarly to turn (left) and descend the pole (right). Male and female mice performed similarly, and no genotype effects were present. Values are expressed as means ± SEM. n=6–7 per group.

Spatial learning and memory is disrupted in Sepp1−/− mice fed a standard diet. (A) Average escape latency per training day over time was measured in the Morris Water Maze. An interaction between Sepp1 genotype and training was present (*p<0.05) and Sepp1−/− mice had significantly longer latency on days 7 and 8 (*p<0.05). (B) In the probe trial, Sepp1−/− spent more time exploring the opposite (OP) quadrant (*p<0.05) and we found a significant interaction effect (genotype x quadrant *p<0.05). They also had non-significant trends toward fewer platform crossings (p=0.0557) (C) and a reduced swim speed (p>0.05) (D). Values are expressed as means ± SEM. n=8–10 per group.

Spatial learning and memory is not disrupted in Scly−/− mice fed a standard diet. (A) Average escape latency per training day over time was measured in the Morris Water Maze. Scly−/− and control mice showed strongly reduced latency over time, and no interaction between genotype and training was apparent. (B) In the probe trial at the end of training, Scly−/− and control mice spent equal time exploring the target (TQ), left (LA) and right adjacent (RA), and the opposite (OP) quadrants (p>0.05). Both genotypes had similar platform crossings (C) and swim speed (D) (p>0.05). Values are expressed as means ± SEM. n=11–13 per group.

Spatial learning is mildly impaired in Scly−/− mice fed a low Se diet. (A) Average escape latency per training day over time was measured in the Morris Water Maze. An interaction between Scly genotype and training was present (*p<0.05), and Scly−/− mice had significantly longer latency on days 2, 3, 5 and 6 (*p<0.05). (B) In the probe trial at the end of training, Scly−/− and control mice spent equal time exploring the target (TQ), adjacent (LA, RA) and opposite (OP) quadrants (p>0.05). Both genotypes had similar platform crossings (C) and swim speed (D) (p>0.05) in the probe trial. Values are expressed as means ± SEM. n=13 per group.

Expression of selenoproteins is decreased in Se-deficient Scly−/− mice brains. (A) GPX1, (B) GPX4, and (C) Sepw1 protein was measured by western blot and quantified by integrated intensity (n=8). Below each graph is a representative sample of the blot including loading equivalence determined by tubulin. GPX1, GPX4, and Sepw1 were drastically reduced in Scly−/− mice (***p<0.0001). All selenoprotein values are normalized to relative amounts of tubulin, and expressed as means ± SEM.

Glutathione peroxidase activity is decreased in Se-deficient Scly−/− mice brains. Total GPX activity in brain (n=8) (A), liver (n=8) (B), and serum (n=12–14) (C) was assayed with a coupled reaction measuring NADPH oxidation. Mice were fed the low Se diet from weaning until time of sacrifice, when tissues were harvested for analysis. Although both brain and liver GPX activity were reduced by approximately half (***p<0.001), serum GPX activity was not significantly reduced. Values are standardized to total protein concentration (brain, liver) or volume of serum and expressed as means ± SEM.